Microbial & Biochemical Technology
Ghaly et al., J Microb Biochem Technol 2013, 5:4
http://dx.doi.org/10.4172/1948-5948.1000110
Research
Article
Review Article
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Access
Fish Processing Wastes as a Potential Source of Proteins, Amino Acids
and Oils: A Critical Review
Ghaly AE*, Ramakrishnan VV, Brooks MS, Budge SM and Dave D
Department of Process Engineering and Applied Science, Faculty of Engineering Dalhousie University, Halifax, Nova Scotia, Canada
Abstract
The fish processing industry is a major exporter of seafood and marine products in many countries. About 70%
of the fish is processed before final sale. Processing of fish involves stunning, grading, slime removal, deheading,
washing, scaling, gutting, cutting of fins, meat bone separation and steaks and fillets. During these steps significant
amount of waste (20-80% depending upon the level of processing and type of fish) is generated which can be utilized
as fish silage, fishmeal and fish sauce. Fish waste can also be used for production of various value added products
such as proteins, oil, amino acids, minerals, enzymes, bioactive peptides, collagen and gelatin. The fish proteins
are found in all parts of the fish. There are three types of proteins in fish: structural proteins, sacroplasmic proteins
and connective tissue proteins. The fish proteins can be extracted by chemical and enzymatic process. In the
chemical method, salts (NaCl and LiCl) and solvents (isopropanol and aezotropic isopropanol) are used, whereas
during the enzymatic extraction, enzymes (alcalase, neutrase, protex, protemax and flavorzyme) are used to extract
proteins from fish. These fish proteins can be used as a functional ingredient in many food items because of their
properties (water holding capacity, oil absorption, gelling activity, foaming capacity and emulsifying properties). They
can also be used as milk replacers, bakery substitutes, soups and infant formulas. The amino acids are the building
blocks of protein. There are 16-18 amino acids present in fish proteins. The amino acids can be produced from fish
protein by enzymatic or chemical processes. The enzymatic hydrolysis involves the use of direct protein substrates
and enzymes such as alcalase, neutrase, carboxypeptidase, chymotrypsin, pepsin and trypsin. In the chemical
hydrolysis process, acid or alkali is used for the breakdown of protein to extract amino acids. The main disadvantage
of this method is the complete destruction of tryptophan and cysteine and partial destruction of tyrosine, serine and
threonine. The amino acids present in the fish can be utilized in animal feed in the form of fishmeal and sauce or
can be used in the production of various pharmaceuticals. The fish oil contains two important polyunsaturated fatty
acids called EPA and DHA or otherwise called as omega-3 fatty acids. These omega-3 fatty acids have beneficial
bioactivities including prevention of atherosclerosis, protection against maniac–depressive illness and various other
medicinal properties. Fish oil can also be converted to non-toxic, biodegradable, environment friendly biodiesel using
chemical or enzymatic transesterification.
Keywords: Fish; Fish processing; Fish waste; Storage; Slime; Rigor
mortis; Autolysis; Protein; Amino acids; Collagen; Oil; Enzymes;
Silage; Enzymatic hydrolysis; Enzyme membrane reactor
Introduction
The fish processing industry in Canada is amajor exporter of
seafood and marine products. Canada exports 75% of its fish products
to more than 80 countries. In the year 2012, exports from Canada
amounted to 595,615.738 metric tonnes of fish worth $4.15 billion [1].
Canada has the world’s longest coastline (244,000 km) which makes
25% of the entire world's coastline. Atlantic Canada represents 40,000
km of coastline which comprises four major provinces. It exports high
quality harvested ground fish, shellfish and pelagic fish accounting
for 85% of the total Canadian fish landings [2]. The Pacific fishery
accounts for 14% of the total fish landings and includes cod, redfish
sp., flatfish, hake, herring, tuna, salmon and calms. The freshwater
fishery accounted for 1% of the total landings and includes perch, pike,
whitefish, yellow pickerel and smelt. The aquaculture production in
Canada for the year 2011 reached 161,036 tonnes worth $845,598 [3].
The world marine capture fisheries contribute more than 50% of
the total world fish production. About 70% of fish is processed before
final sale, resulting in 20-80% of fish waste depending on the level
of processing and type of fish [4]. In addition, a significant amount
of the total catch from fish farming is discarded each year. Also, fish
processing operation require large volumes of potable water which
results in significant amounts of waste water [5]. The majority of fish
wastes are disposed of in the ocean. The aerobic bacteria present in the
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
water breakdown the organic matter in the presence of oxygen leading
to a considerable reduction of oxygen in water. There are also overloads
of nitrogen, phosphorous and ammonia, which lead to pH variation,
increased turbidity of the water and as a result of the decomposition
of algae. The reduction in water oxygen content creates an anaerobic
condition that leads to the release of foul gases such as hydrogen sulfide
and ammonia, organic acids and greenhouse gases such as carbon
dioxide and methane [6].
The discards from the processing plants amount to 20 million
tonnes which is equivalent to 25% of the world’s total production
from marine capture fisheries [4]. These wastes can be used to produce
fish protein concentrate, fish oils and enzymes (such as pepsin and
chymotrypsin) as well as other value added products. The fish oil is
used for products such as margarine, omega-3 fatty acids and biodiesel.
*Corresponding author: Dr. Abdel Ghaly, Department of Process Engineering
and Applied Science, Faculty of Engineering, Dalhousie University, Halifax, Nova
Scotia, Canada, Tel: (902)494-6014; E-mail: [email protected]
Received September 25, 2013; Accepted November 09, 2013; Published
November 15, 2013
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013)
Fish Processing Wastes as a Potential Source of Proteins, Amino Acids and
Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/19485948.1000110
Copyright: © 2013 Ghaly AE, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited
Volume 5(4): 107-129 (2013) - 107
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
The fish protein concentrate is used as human food and animal feed.
Fish protein is also rich in amino acids which are highly suitable for
human consumption [7].
Fish Production
World fish production
All around the world, fish is produced from capture fisheries and
aquaculture [3,8]. About 154 million tonnes of fish were produced in
the year 2011 (Table 1) with a value of $217.5 billion. Approximately
131 (85%) million tonnes were directly utilized as food and the rest
(15%) was underutilized as live bait for fishing, ornamental products
(pearls and shells), feed for carnivorous farmed species and marine
worm. There has been a sustained growth in the fish supply during the
last 50 years with an average growth rate of 3.2% each year which is
higher than the growth rate of world's population (1.7%). Therefore,
the per capita fish supply increased by 17.5% over 10 year period as
shown in Table 2. Table 3 shows the top ten fish harvesting countries in
the world. The production of fish in China, Indonesia, India and Russia
has increased while fish production decreased in other countries over
the ten year period. Fish and fishery products are important sources of
protein and essential micronutrients. In 2009, fish accounted for 16.6%
of the world’s intake of animal protein and 6.5% of all the protein
available in the world [9].
The world's capture fish production during a ten year period (20002011) is shown in Table 1. The total fish capture in 2011 was 90.4 million
tonnes or about 58.7% of the total fish production. The inland capture
fish production increased by 30.68% whereas the marine capture fish
landings decreased by 9.1% which resulted in a net reduction of 5.43%
in the total fish capture [9].
Currently, aquaculture accounts for 40.33% of the world's fish
production. It is defined as the farming of fish, shellfish and aquatic
plants in fresh or saltwater [3]. Aquaculture has grown to 63.6 million
tonnes in 2011. Around 600 aquatic species are raised in captivity
worldwide. The Americas accounted for 4.30%, Europe accounted for
Production
2000
2001
2002
2003
4.2%, Africa accounted for 2.2% and Oceania accounted for 0.30% of
the world aquaculture production in the year 2010. However, Asia
accounted for the majority (89%) of world aquaculture production in
2010. The Asian aquaculture fish production comprises of fin fishes
(64.6%), molluscus (24.2%), crustaceans (9.7%) and other species
(1.5%). The aquaculture production by region for the year 2010 is
shown in Table 4. The top ten aquaculture producers of the world in
the year 2010 are shown in Table 5 [9].
Canada fish production
In Canada, fish are produced by both commercial fisheries (fresh
and sea water) which include capture fisheries and aquaculture.
The total landing from sea fisheries in the year 2011 was 850,533
metric tonnes with a value of $2,107,402. The Atlantic Region accounted
for 703,905 metric tonnes (82.76%) with a value of $1,828,714 and the
Pacific region accounted for 146,628 metric tonnes (17.24%) with a
value of $278,688. The Sea fish production has decreased by 15.24% in
the year 2011 when compared to the production in the year 2000. The
fresh water fisheries contributed another 25,744 tonnes with a value of
$58,206. The amounts of fish produced from sea water and fresh water
during the period of 2000-2011 are shown in Figure 1. The amount of
fish produced from fresh water fisheries gradually decreased by 36.69%
over the 10 year period. On the other hand, the production from sea
fisheries increased reaching its highest production (1,176,229 tonnes)
in 2004 and gradually decreased reaching (850,553 tons) in 2011, a net
loss of 152,967 tonnes (15.24%) compared to that of 2000 [8].
There are different types of aquaculture operations depending
upon the species, environment and culture technologies used as
shown in Figure 2 [10]. Aquaculture in Canada is carried out in all
Canadian Provinces and in the Yukon Territory. Most of the east and
west coasts comprise of finfish and shellfish. Trout are found in fresh
water aquaculture in all provinces. Finfish aquaculture in Canada also
includes active tilapia, sturgeon, Atlantic halibut and other operations.
The fish production from aquaculture during the period of 2000-2011
2004
2005
2006
2007
2008
2009
2010
2011
CAPTURE
Inland
8.8
8.9
8.7
9.0
8.6
9.4
9.8
10.0
10.2
10.1
11.2
11.5
Marine
86.8
84.2
84.5
81.5
83.8
82.7
80.2
80.4
79.5
79.2
77.4
78.9
TOTAL
95.6
93.1
93.2
90.5
92.4
92.1
90.0
90.3
89.7
89.6
88.6
90.4
AQUACULTURE
Inland
21.2
22.5
24.0
25.5
25.2
26.8
31.3
33.4
36.0
38.1
41.7
44.3
Marine
14.3
15.4
16.4
17.2
16.7
17.5
16.0
16.6
16.9
17.6
18.1
19.3
Total
35.5
37.9
40.4
42.7
41.9
44.3
47.3
49.9
52.9
55.7
59.9
63.6
TOTAL WORLD FISHERIES
131.1
131.0
133.6
133.2
134.3
136.4
137.3
140.2
142.6
145.3
148.5
154.0
Table 1: World fish production in million tonnes from 2000 to 2011 [9].
Production
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
Human consumption
96.9
99.7
100.7
103.4
104.4
107.3
114.3
117.3
119.7
123.6
128.3
130.8
Non-food uses*
34.2
31.3
32.9
29.8
29.8
29.1
23.0
23.0
22.9
21.8
20.2
23.2
Total fish supply
131.1
131.0
133.6
133.2
134.2
136.4
137.3
140.3
142.6
145.4
150.1
154.0
Population (billions)
6.1
6.1
6.3
6.4
6.4
6.5
6.6
6.7
6.7
6.8
6.9
7.0
Per capita food fish supply (kg)
16.0
16.2
16.0
16.3
16.2
16.5
17.4
17.6
17.8
18.1
18.6
18.8
*Live bait for fishing
Live ornamental species
Ornamental products (pearls and shells)
Feed for carnivorous farmed species
Culture of biomass for palanton, Artemia and marine worm
Table 2: World fish utilization in million tonnes from 2000 to 2011 [9].
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
Volume 5(4): 107-129 (2013) - 108
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
Country
2001
2010
China
14,403,875.0
15,665,587.0
Difference (%)
8.75
Indonesia
4,294,458.0
5,384,418.0
25.38
India
3,817,092.0
4,694,970.0
22.99
USA
4,981,800.9
4,378,683.7
-12.10
Peru
7,991,712.0
4,265,459.0
-46.62
Japan
4,837,830.2
4,141,312.4
-14.39
Russia
3,638,827.0
4,075,541.0
12.00
Myanmar
1,187,880.0
3,063,210.0
157.87
Chile
4,031,799.0
3,048,316.0
-24.39
Norway
2,862,152.0
2,675,292.0
-6.52
Other Countries
39,935,214.0
38,110,903.7
-4.56
World Total
91,982,640.1
89,503,692.8
-2.69
Table 3: Top ten capture fish producers of the world in metric tonnes [8].
Regions
Africa
2000
2010
Tonnes
(%)
Tonnes
(%)
399,676
1.20
1,288,320
2.20
Americas
1,423,433
4.40
2,576,428
4.30
Asia
28,422,189
87.70
53,301,157
89.00
Europe
2,050,958
6.30
2,523,179
4.20
Oceania
121,482
0.40
183,516
World
32,417,738
Figure 2: Different types of aquaculture in Canada [10].
100.00
59,872,600
0.30
100.00
Table 4: World Aquaculture production by region from 1970 to 2010 [9].
World
Tonnes
(%)
China
36,734,215
61.35
India
4,648,851
7.76
Vietnam
2,671,800
4.46
Indonesia
2,304,828
3.85
Bangladesh
1,308,515
2.19
Thailand
1,286,122
2.15
Norway
1,008,010
1.68
Egypt
919,585
1.54
Myanmar
850,697
1.42
Philippines
744,695
1.24
7,395,281
12.35
59,872,600
100.00
Other
Total
Table 5: Top ten aquaculture producers in the world in 2010 [9].
Figure 3: Canadian aquaculture production from 2000-2011 [3].
is shown in Figure 3. In 2011, the total production of fish from aqua
culture was 163,036 tonnes with a value of $845,598. In 2010, British
Columbia, New Brunswick produced and Newfoundland and Labrador
produced 58%, 18% and 13% of the total aquaculture production,
respectively [3].
Fish processing
Most fish processing plants process fish using the following
steps: stunning of fish, grading, removal of slime, scaling, washing,
deheading, gutting, cutting of fins, slicing into steaks, filleting, meatbone separation, packaging, labelling and distribution (Figure 4). The
amount of waste collected from each Canadian province is shown in
Table 6.
Stunning
The stunning of fish is the first and most critical step in the
processing of fresh water and farmed fish because prolonged agony
experienced by the fish causes the production of undesired substances
in fish tissues. The oxygen deficiency in the blood and muscle causes
accumulation of lactic acid and leads to paralysis of the neural system.
Stunning of the fish produces enough movements to break the vertebrae
and rupture blood vessels. Red spots appear on the surface of the skin
and in the muscle tissues near the backbone [11].
Figure 1: Canadian fish production from 2000-2011 [8].
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
Erikson et al. [12] stated that some of the wild fish are subjected
to asphyxiation on board after capture until they die using CO2. Robb
et al. [13] reported that immersion in CO2 saturated water resulted
in narcoss, with a loss of brain function. During the initiation of
Volume 5(4): 107-129 (2013) - 109
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
and increased quality of fish products at the end of the processing chain
[16,20].
Slime removal
Fish secretes slime on its surface as a protection mechanism against
harmful conditions. The slime secretion stops before rigor mortis.
Pseudomonas species are one of the potent spoilers, always present in
the sea water, and fish slime provides them with a perfect environment
to grow [21,22]. Anaerobic bacteria present during fish processing can
produce hydrogen sulfide by taking up sulfur compounds from the
slime, skin and flesh [23,24]. Therefore, the slime should be removed by
continuous washing. Slime present in some of the species such as eel,
trout and other fresh water species should be soaked in a solution of 2%
baking soda and then washed in a cylindrical rotating washer [16,22].
Figure 4: Fish processing steps.
Landing
Province
Product
Scaling
Waste
(Tonnes)
(%)
(Tonnes)
(%)
(Tonnes)
(%)
New Brunswick
113588
13.95
89012
78.36
24576
21.63
New Foundland and
Labrador
267959
32.92
120999
45.15
146960
54.84
Nova Scotia
366381
45.01
146708
40.04
219673
59.95
Prince Edward Island
66046
8.11
39000
59.04
27046
40.95
Total
813974
100.00 395719
48.61
418255
51.38
Table 6: Fish waste amount by province in 2001 [4].
narcosis, the fish shows strong aversion lasting from 30sec to 3min
after immersion in CO2. The immersion causes pH imbalances in the
blood and thereby disrupting the function of brain. Poli et al. [14]
reported that the CO2 narcosis initiated greater lactic acid production.
Roth et al. [15] reported that CO2 stunning of fish is the most stressful
method causing panic and flight reactions due to acidic and hypoxic
environment, which leads to quick depletion of energy phosphogens
and increases the earlier onset of rigor mortis.
In some cases of farmed fish, the fish is directly plunged into
iced water in which the temperature is kept close to 0°C. It is critical
that the temperature is kept low because the fish would not die due
to temperature shock but by asphyxia which affects the quality and
texture of the fish [16].
Borderias and Sanchez-Alonso [16] stated that electrical stunning
is recommended for killing salmon and grass carp over CO2 because it
causes earlier onset of rigor mortis and fast eradenosine triphosphate
(ATP) stunning carried out on turbot (Scopthalmus maximus) resulted
in a rapid drop in the pH and potential increase in fillet gaping. Erikson
et al. [12] stated that the efficiency of electrical stunning depended on
whether the fish is stunned in the head or through the whole body.
Morzel et al. [17] reported that during stunning, the initial pH was
low and rapid onset of rigor mortis occurred resulting in flesh that
was softer, redder and darker when compared to fish cut by percussion
or bleeding. Roth et al. [15] reported that when Atlantic salmon were
electrically stunned in water for 1.5 s, there was no accelerated rigor
development and no injuries were observed in the fish.
Grading
The second step in fish processing is fish grading by species and size.
Grading of fish can be done manually or by mechanical equipment.
The mechanical grading is more precise for fish before or after rigor
mortis than for fish in a state of rigor mortis. The automated grading
instruments are 6-10 times more efficient than manual grading [18,19].
The basic benefits of the automated system are: low production costs
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
The process of scaling is one of the toughest steps in fish processing
and is extremely labour intensive. The scales may harbour bacterial
pathogens and removing them will keep the fish fresh while refrigerated
or frozen [25,26]. The scaling can be done manually with a hard brush
or scaling blades. The scales of some fish such as perch, pike-perch,
carp and bream are difficult to remove. These fish are first blanched
in boiling water for 3-6 seconds and then scaled using mechanized
hand-held scalers in a motion perpendicular to the long body axis. The
electrical scalers are more efficient in completely eliminating scales
than the manual tools and save lot of time [16].
Washing
The primary goal of washing is to clean and remove the accumulated
bacteria on the fish. The effective washing of fish depends upon the
fish:water ratio, the quality of water and kinetic energy of the water
stream. The recommended washing fish:water ratio is 1:1. However,
the amount of water used increases by two fold during processing. Use
of potable water is recommended during freshwater fish processing
[16]. Washing is carried out using vertical drum, horizontal drum and
conveyor belt washers. The washing time is about 1-2 min and these
mechanized washers can be used to process whole fish, deheaded and
gutted fish as well as fish fillets. Washing action does not cause any
physical damage to the product [27]. The washing process is always
continuous and is accomplished by spraying pressurized water. The
dirty water is collected in the waste basins. The amount of wastewater
produced during each step in fish processing is shown in Tables 7-9
[28].
Deheading
The fish head constitutes up to 20% of its weight (Table 10) and it
is usually considered as an inedible part [29]. The fish can be deheaded
manually or mechanically. Manual cutting is easier for small fresh
water fish. Larger fish ranging from 20-40 cm can be deheaded using
mechanical devices. Fish can be cut in three different ways: round cut,
straight cut and contoured cut. In most fish plants, manual deheading
is performed because it causes minimal flesh loss. A cut around the
operculum is a called a round cut and it results in the lowest meat loss.
The contour cut runs perpendicular to the fish backbone and then at an
angle of 45°. This cut is mainly used when the final product is a boneless
and skinless fillet [16]. Machines with a guillotine cutter are suitable
for larger fish under-going round or contour cuts. Machines with a
manually-operated circular saw are suitable for larger fish undergoing
straight cuts. The amount of deheaded waste produced from fish
processing is 27-32% as shown in Table 7 [28].
Volume 5(4): 107-129 (2013) - 110
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
Inputs
Outputs
Process
Fish
(kg)
Energy
(kW h)
Wastewater
(m3)
BOD
(kg)
COD5
(kg)
Nitrogen
(kg N)
Phosphorous
(kg P)
Solid waste
(kg)
White fish filleting
1000
Ice: 10-12
Freezing: 50-70
Filleting: 5
5-11
35
50
-
-
Skin: 40-50
Heads: 210-250
Bones: 240-340
Oily fish filleting
1000
Ice: 10-12
Freezing: 50-70
Filleting: 2-5
5-8
50
85
2.5
0.1-0.3
400-450
Frozen fish thawing
1000
-
5
-
1-7
-
-
-
De-icing and washing
1000
0.8-1.2
1
-
0.7-4.9
-
-
0-20
Grinding
1000
0.1-0.3
0.3-0.4
-
0.4-1.7
-
-
0-20
Scaling of white fish
1000
0.1-0.3
10-15
-
-
-
-
Scales: 20-40
Deheading of white fish
1000
0.3-0.8
1
-
2-4
-
-
Head and debris: 270-320
Filleting of deheaded white fish
1000
1.8
1-3
-
4-12
-
-
Frames and off cuts: 200-300
Filleting of ungutted oily fish
1000
0.7-2.2
1-2
-
7-15
-
-
Entrails, tails, heads and frames:
400
Skinning white fish
1000
0.4-0.9
0.2-0.6
-
1.7-5
-
-
Skin: 40
Skinning oily fish
1000
0.2-0.4
0.2-0.9
-
3-5
-
-
Skin: 40
Trimming and cutting of white
fish
1000
0.3-3
0.1
-
-
-
-
-
Packaging of fillets
1000
5-7.5
-
-
-
-
-
-
Freezing and storage
1000
10-14
-
-
-
-
-
-
Handling and storage of fish
1000
10-12
-
-
130-140
-
-
-
Unloading of fish
1000
3
2-5
-
27-34
-
-
-
Table 7: Inputs and outputs of fish production processing [28].
Process
Inputs
Outputs
Fish
(kg)
Energy
(kW h)
Wastewater
(m3)
BOD
(kg)
COD5
(kg)
Nitrogen
(kg N)
Phosphorous
(kg P)
Solid waste
(kg)
Canning
1000
150-190
15
52
116
3
0.1-0.4
Head: 250
Bones: 100-150
Unloading fish for canning
1000
3
2-5
-
27-34
-
-
-
Precooking of fish to be canned
1000
0.3-11
0.07-0.27
-
-
-
-
Inedible parts: 150
Nobbing and packing in cans
1000
0.4-1.5
0.2-0.9
-
7-15
-
-
Head and entrails: 150
Bones and meat: 100-150
Draining of cans containing precooked fish
1000
0.3
0.1-0.2
-
3-10
-
-
-
Sauce filling
1000
-
-
-
-
-
-
Spillage of sauce and oil: varies
Can sealing
1000
5-6
-
-
-
-
-
-
Washing of cans
1000
7
0.04
-
-
-
-
-
Sterilization of cans
1000
230
3-7
-
-
-
-
-
Table 8: Inputs and outputs of fish canning process [28].
Process
Inputs
Outputs
Fish
(kg)
Energy
(kW h)
Wastewater
(m3)
BOD
(kg)
COD5
(kg)
Nitrogen
(kg N)
Phosphorous
(kg P)
Solid waste
(kg)
Fish meal and fish oil
1000
Electricity: 32
-
-
-
-
-
-
Cooking of fish
1000
90
-
-
-
-
-
-
Pressing the cooked fish
1000
-
750kg water
150kg oil
-
-
-
-
Press cake: 100 dry matter
Drying of press cake
1000
340.0
-
-
-
-
-
-
Fish oil polishing
1000
Hot water
0.05-0.1
-
5
-
-
-
Stick water evaporation
1000
475
-
-
-
-
-
Concentrated stick water: 250
Dry matter: 50
Table 9: Inputs and outputs of fish meal production process [28].
Gutting
Gutting of the fish is the removal of internal organs and optionally
cleaning the body cavity of the peritoneum, kidney tissue and blood. In
the gutting process, the fish is cut longitudinally to remove the internal
organs on a table made of special material which is easy to wash and
does not absorb fluids. The table is rinsed and periodically disinfected.
There are some mechanical gutting machines used for trout, eel and
J Microb Biochem Technol
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other fish, but their use increases the fish processing cost [30]. The
internal organs constitutes around 5-8% of the fish weight [29]. The
amount of waste in the gutting processing is shown in Table 7 [28].
Cutting of fins
Fins constitute around 1-2% of the fish weight. The amount of
fin waste after fish processing is shown in Table 7 [28]. Fins are cut
Volume 5(4): 107-129 (2013) - 111
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
Component
Average Weight (%)
Head
21
Gut
7
Liver
5
Roe
4
Backbone
14
Fins and lungs
10
Skin
3
Fillet, skinned
36
Table 10: Average composition of fish [29].
manually either by a knife or by mechanized rotating disc knives [31].
This process is mostly carried out after deheading and gutting. This
process is difficult for cutting larger fish. The mechanical knives are
provided with a slit opening in which the fins are cut when the fish are
passed through it manually [16,32].
Steaks and fillets
Fillets are pieces of meat containing only the dorsal and abdominal
muscles. The fillets are processed manually or mechanically. Manual
filleting is carried out in small fresh water fish industries and
mechanical filleting is used for processing marine fish. Deheaded whole
fish are sliced into steaks by cutting perpendicular to the backbone.
Small and medium-sized fish are cut manually in a concave basin with
evenly-spaced slots to facilitate slicing. The average thickness of the fish
pieces is 2.5-4.5 cm. Large fish such as cyprinids are sliced mechanically
because of their solid and massive backbone. These pieces are more
popular in the retail market and the canning industry [16,33]. Once the
fillet leaves the filleting stations, three products remain: napes, block
and trimmed fillet. Napes are the thinnest part of a fillet that covered
the guts before the fish was gutted. Blocks are the parts removed from
fillets for aesthetic purposes. Trimmed fillet is the final product in
which the napes and pin bone attached to some fillets will be removed
[30].
Meat bone separation
Around 30-50% of the meat is usually left along the ribs and
backbone during filleting. In smaller fish, the loss of meat is high and so
minced fish meat is gaining more attention [34,35]. Minced fish meat
can also be produced from less valuable species after deheading and
carefully removing their internal organs. In this process, the meat is
removed from skin, scales and bones through automated separators.
The fish travels along a conveyor belt which runs closely to a perforated
cylinder. The meat is squeezed through the holes due to the pressure
applied from the conveyer belt and the bones are scraped away [16]. The
minced fish meat stability is much less than that of intact fish muscle
and so it is frozen immediately. It is used to produce fish burgers, fish
sticks, canned fish, vegetable mixes and fish dumplings [36,37].
Composition of Fish Waste
The composition of the fish varies according to the type of species,
sex, age, nutritional status, time of year and health. Most of the fish
contains 15-30% protein, 0-25% fat and 50-80% moisture [7,38].
Suvanich et al. [39] reported that the composition of catfish, cod,
flounder, mackerel and salmon variedaccording to the species (Table
11). Mackerel had the highest fat content (11.7%) and cod had the
lowest (0.1%). Salmon had the highest protein content (23.5%) and
flounder had the lowest (14%). The moisture content of the five fishes
varied between 69 and 84.6% but the ash content of all species was
similar.
J Microb Biochem Technol
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Solid fish waste consists of head, tails, skin, gut, fins and frames.
These by products of the fish processing industry can be a great source
of value added products such as proteins and amino acids, collagen
and gelatin, oil and enzymes as shown in Table 12 [40,41]. These wastes
contain proteins (58%), ether extract or fat (19%) and minerals. Also,
monosaturated acids, palmitic acid and oleic acid are abundant in fish
waste (22%).
Proteins
Fish frames contain significant amounts of muscle proteins. These
muscle proteins are highly nutritious and easily digestible. Therefore,
proteins from this part of the fish waste can be extracted by enzymatic
hydrolysis rather than being discarded as waste [42]. Proteins derived
from fish are nutritionally superior when compared to those of plant
sources. They have a better balance of the dietary essential amino
acids compared to all other animal protein sources [43,44]. However,
fish muscle proteins are more heat sensitive than mammalian muscle
proteins [45]. Also, fish muscle proteins from the cold water species are
more susceptible to denaturation by heat when compared to those of
tropical water fish. The T-50 values (the temperature required for 50%
denaturation of the fish muscles) are influenced by the pH and were
reported to be in the range of 29-35°C at a pH of 7.0 and in the range of
11-27°C at a pH of 5.5 [46].
Fish muscles consist of two types: light and dark. The proportion of
dark muscle is low in white fish such as cod and haddock where there
is a small strip of dark or red muscle just under the skin on both sides
of the body. In fatty fish, such as herring and mackerel, the percentage
of dark muscles is high and the muscles contain more vitamins and fats
as shown in Figure 5 [7]. Light muscle is more abundant and contains
about 18-23% proteins.
About 70-80% of the fish muscles are made up of structural proteins
and the remaining 20-30% are composed of sarcoplasmic proteins with
about 2-3% insoluble connective tissue proteins. Myofibrillar proteins
Fish Type
Fat (%)
Ash (%)
Protein (%)
Catfish
7.7
0.9
15.4
Moisture (%)
76.3
Cod
0.1
1.1
18.2
80.8
Flounder
0.7
1.3
14.0
84.6
Mackerel
11.7
1.1
18.8
69.0
Salmon
1.6
1.1
23.5
74.3
Table 11: Composition of the fish fillets determined by standard methods [39].
Nutrient
Fish waste
Crude protein (%)
57.92 ± 5.26
Fat (%)
19.10 ± 6.06
Crude fiber (%)
1.19 ± 1.21
Ash (%)
21.79 ± 3.52
Calcium (%)
5.80 ± 1.35
Phosphorous (%)
2.04 ± 0.64
Potassium (%)
0.68 ± 0.11
Sodium (%)
0.61 ± 0.08
Magnesium (%)
0.17 ± 0.04
Iron (ppm)
100.00 ± 42.00
Zinc (ppm)
62.00 ± 12.00
Manganese (ppm)
6.00 ± 7.00
Copper (ppm)
1.00 ± 1.00
Values in % or mg/kg (ppm) on a dry matter basis.
Table 12: Composition of fish waste [40].
Volume 5(4): 107-129 (2013) - 112
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
Collagen and gelatin
The fish skin waste is a good source for collagen and gelatin
which are currently used in food, cosmetic and biomedical industries.
Collagen and gelatin are two different forms of same macromolecule in
which gelatin is a partially hydrolysed form of collagen. The collagen
and gelatin are two unique and more significant forms of proteins in
comparison to that of fish muscle proteins.
Figure 5: Types of muscles in white fish and fatty fish [7].
are the primary food proteins and they make up about 66-77% of the
total protein content in the fish meat. These myofibrillar proteins
comprise of 50-60% myosin and 15-30% actin [47]. The myosin fibers
can be cleaved by proteases trypsin and chymotrypsin on one end
and on the other end with papain. During this cleavage, the myosin
fibers are divided into heavy meromyosin and light meromyosin with
different functional properties. Actin occurs in two forms, G-actin, a
spherical monomer and F-actin, a large polymer which connects to
myosin [46].
Amino acids
Fish protein contains a well balanced amino acid composition.
Fish is composed of 16-18 amino acids based upon the species type
and seasonal variations. The amino acid profile of Atlantic mackerel
is shown in Table 13 [48,49]. Fish contains well balanced amino acid
compositions consisting of eight essential amino acids and eight nonessential amino acids. Due to the rich amino acid content of fish, it is
being utilized as fish meal, fish sauce, fertilizer, animal feed and fish
silage [50].
Oil
Fish processing by products contain fish oil. The amount generally
depends upon the fat content of the specific fish species. Generally,
fish contains 2-30% fat. Almost 50% of the body weight generated as
waste during the fish processing would be a great potential source for
good quality fish oil which can be used for human consumption or
production of biodiesel. The fish oil consists of two main fatty acids,
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These
two fatty acids are polyunsaturated fatty acids and are classified as
omega-3 fatty acids. They are mainly found in the marine animals
which have high polyunsaturated fatty acid content [51].
Bioactive peptides
Proteins extracted from the fish muscle contain a number of
peptides which have manybioactivities such as antihypertensive,
antithrombotic, immune modulatory and antioxidative properties
[52]. The bioactive peptides obtained from the fish muscle have
anticoagulant and antiplatelet properties, which are the main reason
behind the capability of peptides obtained from the fish to inhibit
coagulation factors in the intrinsic pathway of coagulation [53].
The protein obtained by the enzymatic hydrolysis of the fish muscle
has several nutritional and functional properties from which many
biologically active peptides can be obtained [54].
J Microb Biochem Technol
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The significance lies upon the amino acid content; more than 80%
are non-polar amino acids such as glycine, alanine, valine and proline
[55]. Heat denaturation of collagen easily converts it into gelatin. The
collagen and gelatin extracted from bovine sources pose the risk of
mad cow disease or bovine spongiform encephalopathy (BSE), whereas
the collagen and gelatin extracted from fish skin eliminates these risks
of BSE. The gelatin extracted enzymatically from fish skin has better
biological activities as antioxidants and antihypertensive agents. The
gelatin has a unique repeating sequence of glycine-proline-alanine
in their structure compared to the peptides derived from fish muscle
protein and it is the main reason behind the antioxidative propertyof
gelatin [56,57].
Enzymes
The internal organs of the fish are a rich source of enzymes, many of
which exhibit high catalytic activities at relatively low concentrations.
The enzymes which are available in fish include: pepsin, trysin,
chymotrypsin and collagenase. These enzymes are commercially
extracted from the fish viscera in a large scale. They possess better
catalytic properties, good efficiency at lower temperatures, lower
sensitivity to substrate concentrations and greater stability in a wide
range of pH [56-58].
Pepsin is a proteolytic enzyme which is found in the stomach of fish
and constitutes 5% of the fish weight. It is used in various extraction
processes such as extraction of collagen gelatin and can be used as
rennet substitute and can be used to digest proteins. The optimum
conditions for pepsin were pH 2-4 and temperature of 30°C [59-63].
Chymotrypsin is an endoprotease which hydrolyze proteins by
breaking the central peptide bonds, and yielding a mixture of peptides
and amino acids. Fish chymotrypsin consists of two different forms:
Amino acid
Mackerel Fish
Whole Tissue
White Muscle
Dark Muscle
Aspartic acid
11.8
12.2
11.4
Threonine
5.7
5.5
5.5
Serine
4.5
5.2
4.1
Glutamic acid
15.8
18.0
15.6
Proline
1.5
4.6
1.5
Glycine
6.0
6.2
4.6
Alanine
7.7
7.3
7.3
Valine
7.8
6.8
8.5
Methionine
2.7
4.6
2.8
Isoleucine
5.5
6.0
5.6
Leucine
9.4
10.0
8.8
Tyrosine
3.5
3.9
3.4
Phenylalanine
4.2
4.0
3.1
Histidine
4.5
3.8
5.2
Lysine
7.9
8.0
7.6
Arginine
7.6
5.9
7.1
Table 13: Amino acid profile of Atlantic Mackerel (g amino acid/100 g protein) [48].
Volume 5(4): 107-129 (2013) - 113
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
chymotrypsin A and chymotrypsin B. Fish chymotrypsin has higher
catalytic activity, lower thermo stability and differs in polypeptide
amino acid composition to bovine chymotrypsin. The optimum
conditions for chymotrypsin was pH 7-8 and temperature of 50°C
[58,64,65].
Trypsin is an important pancreatic serine protease which is
synthesized as proenzyme by pancreatic acinar cells and is secreted to
the intestine. Fish trypsin is similar to mammalian trypsin in molecular
weight and amino acid composition [66]. Trypsin cleaves the peptide
bond on the carboxyl side of arginine and lysine. They hydrolyse
synthetic substrates such as Nα-benzoyl-L-arginine-p-nitroanilide and
tosyl-arginine methyl ester. Trypsin is susceptible to serine-protease
inhibitors such as phenyl-methyl-sulfonyl fluoride (PMSF), soybean
trypsin inhibitor (SBTI) and aprotonin. The optimum conditions for
trypsin isa pH of 9 and a temperature of 40°C [67,68].
Fish collagenases, also called collagenolytic serine proteases which
differ from muscle collagenases, belong to the zinc metalloproteases
and are known to hydrolyse triple type I, II and III tropocollagen
molecule. They show both trypsin and chymotrypsin like activities.
They are most active in the pH range of 6.5-8.0 and at a temperature
of 30°C [67].
Minerals
Fish bones are normally separated after removal of muscle proteins
from the frames. The fish bones account for 30% of the collagen and
are considered an additional source of collagen along with fish skin.
Fish bones contain 60-70% minerals including calcium, phosphorous
and hydroxyapatite [56]. Generally, calcium is deficient in most of the
regular diets and to improve calcium intake, consumption of small
whole fish can be nutritionally valuable. The fish bones obtained from
the fish processing waste can be used to provide calcium. In order
for bones to be a fortified food, they should be converted into edible
form by softening their structure with hot water treatment, hot acetic
acid solutions or by super heated steam cooking [69]. Fish bones are
a very good source of hydroxyapatite (Ca10(PO4)6(OH)2) which can
be used as a bone graft material in medical and dental applications.
Previously autografts, allografts and xenografts were used to solve bone
fractures and damages but they were found to be ineffective due to their
mechanical instability and incompatibility. The important properties
of hydroxyapatite are: it does not break under physiological conditions,
it is thermodynamically stable at physiological pH and it plays an active
role in bone binding [70].
Current Utilization of Spoiled Fish and Fish Waste
Fish silage
Fish silage is an excellent protein source having high biological
properties for animal feeding. Fish silage is a liquid product made from
whole fish or parts of fish that are liquefied by the action of enzymes
in the fish in the presence of added acid. The enzymes present in the
acidic medium breakdown fish proteins into smaller soluble units
while the acid helps to speed up their activity and prevent bacterial
spoilage. Fish silage can be made from spoiled fish, sub-utilized species,
by-products from marine fish, commercial fish waste and industrial
residues from the filleting industry [41,71]. The proteins present in the
fish silage can also be hydrolysed to free amino acids, making the silage
the most available source of amino acids for protein biosynthesis. The
composition of amino acids in the various states of fish silage is shown
in the Table 14 [71].
During fish silage preparation, the raw material is chopped into
small pieces and a 3% by weight solution of 98% formic acid is added
and mixed well and then stored for 48 days. The pH of the mixture
should be less than 4 to prevent bacterial action [72,73]. Fish silage can
also be prepared by a fermentation method in which fish is chopped,
minced and mixed with 5% (w/w) sugar beet molasses. A culture of
Lactobacillus plantarum is inoculated into molasses and incubated
until a population of 107 bacteria per g of molasses is obtained. This
culture is then added in the ratio of 2 ml/kg to the minced fish. The
inoculum is incubated at 30°C for 7 days inside sealed plastic buckets.
Amino acids
SW
FSW
ASW
FW
FFW
AFW
TR
FTR
ATR
Tryptophan
0.79
0.65
0.66
0.97
0.87
1.34
0.52
0.61
0.43
Lysine
10.12
9.16
7.90
7.48
9.92
9.09
9.75
5.94
6.77
Histidine
5.24
5.85
5.70
2.65
3.08
2.75
2.02
2.52
2.20
Arginine
3.03
2.19
6.11
3.62
1.80
7.72
2.46
2.49
7.27
Aspartic acid
9.05
10.79
7.83
10.17
9.62
6.20
10.16
11.79
8.98
Threonine
2.85
4.97
4.58
3.18
5.12
5.28
2.76
4.68
4.72
Serine
2.71
3.23
4.49
3.39
3.52
5.53
2.04
3.72
5.11
Glutamic acid
13.57
14.45
14.04
16.18
13.83
9.26
13.88
14.76
13.10
Proline
3.19
3.66
5.74
4.37
5.57
7.78
7.75
7.22
5.94
Glycine
6.49
5.87
8.17
6.20
6.32
11.55
7.50
9.22
12.32
Alanine
8.60
7.41
7.39
9.27
8.12
6.00
8.81
8.92
7.63
½ Cystine
0.81
0.69
1.54
0.97
1.03
0.63
1.40
0.86
1.34
Valine
6.42
5.77
4.16
5.95
5.83
3.92
6.62
5.06
4.31
Methionine
6.88
6.03
3.75
3.19
4.97
5.31
2.80
5.54
5.37
Isoleucine
5.31
5.05
3.10
5.38
5.00
3.10
6.24
4.63
2.51
Leucine
9.16
8.00
7.33
9.61
9.31
7.57
10.32
6.72
6.23
Tyrosine
1.78
1.90
3.45
2.40
2.02
2.73
1.22
1.70
2.43
Phenylalanine
3.99
4.32
4.08
5.02
4.07
4.26
3.76
3.63
3.35
CP (g/kg)
776.7
596.1
699.1
496.2
420.9
443.8
429.9
358.4
395.9
SW: Commercial Saltwater Fish Waste; FSW: Fermented Saltwater Fish Silage; ASW: Acid Salt Water Fish Silage; FW: Commercial Freshwater Fish Waste; FFW:
Fermented Freshwater Fish Silage; AFW: Acid Freshwater Fish Silage; TR: Tilapia Filleting Residue; FTR: Fermented Tilapia Residue Silage; ATR: Acid Tilapia Residue
Silage; CP: Crude Protein (Dry Matter)
Table 14: Amino acid composition (g/100 g CP) and protein content of fish silage [71].
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
Volume 5(4): 107-129 (2013) - 114
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
The autolysis is later stopped by heating the silage at 90°C for 30 min
[74,75]. Fish silage can be mixed with wheat bran and oven dried at
105°C. Co-drying fish silage with cereals reduces the drying times of
the silage and improves the nutritional content of the silage. To prevent
spoilage of the dried silage it should contain low moisture content.
Water levels greater than 120 g/kg can support bacterial, mould and
yeast growth [76].
According to Gildberg [77], many bioactive products including
peptone, oil and pepsin can be obtained from fish silage. Fish like
Atlantic cod and salmon have high amounts of pepsin in the stomach.
The optimal storage conditions for the recovery of pepsin are pH of 3
and 25°C for 3 days. By ultra-filtration and spray drying, the stomach
silage can provide crude pepsin corresponding to 0.5-1 g of pure pepsin
per kg. The purity of the crude pepsin extracted ranges from 2-10%. The
cod stomach and viscera silage can also provide 100 g low molecular
weight peptone per kg of the raw material.
Fish meal
Fishmeal is a dry powder prepared from whole fish or from fish
filleting wastes which are unacceptable for human consumption. The
raw materials are transported to the processing factories either fresh
or preserved in formaldehyde or sodium nitrate [78]. The production
of fish meal is carried out in six steps: heating, pressing, separation,
evaporation, drying and grinding. When the fish is heated the protein
present in it coagulates and ruptures the fat deposits. This liberates oil
and water. The fish is then pressed which removes large amounts of
liquid from the raw material. The liquid is collected to separate oil from
water. The water which is also known as stick water is evaporated to a
thick syrup containing 30 to 40% solids. Then it is subjected to drying
using press cake method to obtain a stable meal. This meal is grinded to
the desired particle size [79].
Fishmeal obtained from wild-harvested whole fish and shellfish
currently makes up the major aquatic protein source available for
animal feed. The global fishmeal production was 5 million tonnes in
1976 which increased to 7.48 million tonnes in 1994 and then decreased
to 5.74 million tonnes in 2009. In recent years, the fishmeal production
from the fisheries by-products has increased drastically and around 6
million tonnes of fish waste have been used for fishmeal production.
It is estimated that 25% (1.23 million tonnes in 2008) of the total fishmeal produced is from fish by-products [9].
Production and utilization of Fish Protein
Fish proteins are extracted from fish using chemically and
enzymatic methods. Protein hydrolysates obtained from these
processes have various industrial uses such as milk replacers, protein
supplements, stabilizers in beverages and flavour enhancers.
Chemical extraction of fish protein
The most common extraction method used for the fish proteins is
the solvent extraction method. The standard protocol for the solvent
extraction of proteins reported by Sikorski and Naczk [85] is shown
in Figure 6. The whole fish is first ground and the protein is extracted
using isopropanol. After grinding, the supernatant is collected and
extracted three times. The first extraction is carried out at 20-30°C for
50 min in isopropanol. The second extraction is carried out at 75°C for
90 min with isopropanol. The third extraction is carried out at 75°C
for 70 min with azeotropic isopropanol. The final supernatant fraction
is collected, dried, milled and screened to separate out bone particles.
Hermansson et al. [86] reported that the fish protein concentrate
can also be produced at a temperature of 50°C but it will have lower
emulsifying properties and poor solubility. The disadvantages of this
method are poor functionality, off-flavours, high cost of production
and traces of the solvent in the final product, making it commercially
unsuccessful.
Another chemical method for the production of fish protein
concentrate and gelatin (Figure 7) was reported by Arnesen and
Gildberg [87]. 2000 g of the Atlantic cod are added to 2000 ml of
water and the pH is adjusted to 11 with 62 ml of 3 M NaOH. The first
extraction is carried out for 15 min and the sample is centrifuge is then
suspended in 2000 ml and the pH is adjusted to 11 with 3 M NaOH (15
ml). The second extraction is carried out for 60 min and the sample
is then centrifuged. The pellet is again suspended in 2000 ml of water
and the pH is adjusted to 2 with 3M HCl (145 ml). The third extraction
is carried out for 15 min and centrifuged. The supernatants from the
three extracts are pooled together and the pH was adjusted to 7 with
3 M NaOH. The samples are allowed precipitating for 15 min at room
In 1988, around 80% of the total fishmeal produced was used as
feed for pigs and poultry and 10% of the total was used for aquaculture.
Currently, 63% of the fishmeal is being consumed by the aquaculture
industry, 25% by the pigs, 8% by the poultry and 4% by other animals
[80].
Fish sauce
Fish sauce is made from small pelagic fish or by-products using salt
fermentation. Fish are mixed with salt in the ratio of 3:1 at 30°C for six
months and an amber protein solution is drained from the bottom of
the tank. It can be used as a condiment on vegetable dishes and is very
nutritious due to the presence of essential amino acids [81]. Fermented
fish sauce has various biological activities including angiotensin
I-converting enzyme (ACE) inhibitory activity and insulin secretionstimulating activity. Various studies reported ACE inhibitory activity
in the fermented fish sauce from salmon, sardine and anchovy. Three
ACE peptides (gly-trp, ile-trp and val-trp) were found in fermented
fish sauce [82-84].
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
Figure 6: Extraction of fish protein using solvent [85].
Volume 5(4): 107-129 (2013) - 115
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
Kelleher and Hultin [90] used lithium chloride for the extraction
of protein from fish muscle. 40 g of ground muscle were mixed with
760 ml (1:20) of 4.2% LiCl and 0.02 M Li2CO3 at a pH of 7.2 and a
temperature of 25°C. The contents were homogenized in a blender
for 2 min and centrifuged at 2600 g for 20 min. The supernatant was
collected and analyzed for protein using the Biuret method. The same
procedure was used but LiCl was replaced by 5% NaCl with 0.02 M
NaHCO3 and 6% KCl with 0.02 M KHCO3. The results indicated that
lithium chloride was much better than sodium or potassium chlorides
with respect to blending time. Also, lithium chloride had a stable and
consistent protein yield over a wide range of concentrations compared
to the other two salts.
Figure 7: Extraction of muscle proteins and gelatin from Atlantic cod [87].
temperature and the soluble protein is separated by centrifugation
at 4°C for 60 min at 5000 g. Altogether 47.5% of the total protein is
recovered from the pooled extract from muscle and soft tissues. The
solids remaining after the third extraction included bone, skin and
residual muscle tissues.
Batista [88] reported on the extraction of proteins from hake and
monkfish wastes using a chemical method. The minced fish waste was
mixed with water in ratios of 20:1 20:5 for a time ranging from 5-120
min. The pH was in the range of 1-12 and the temperature range was
in the range of 22-55°C. The extraction was carried out with HCl in the
acid phase and with NaOH and Ca(OH)2 in the alkaline phase. After
the extraction is completed, the protein extracts were centrifuged for
15 min at 5000 rpm and the supernatant obtained was filtered through
glass wool. The results indicated that the minimum solubility for hake
waste proteins was seen at a pH in the range of 5-6 for hake fish waste
and at pH of 5 for monkfish waste. The amount of proteins solubilised
at optimum pH was 17% for hake fish waste and 9% for monkfish
waste. The extraction time influenced the amount of protein solubilised
in both hake and monk fish wastes. Higher yield was obtained at 45°C
when NaOH was used for extraction and at 50°C when Ca(OH)2 was
used for extraction. The ratio of 10:1 (fish:water) was found to be more
convenient giving better yield.
Undeland et al. [89] extracted proteins with acid and alkali from
ground whole herring fish (120-300 g) in a blender using 9 volumes of
ice cold distilled water. The proteins present in the homogenate were
solubilized by adding 2 N HCl or 2 N NaOH in drops until a pH of
2.8 or 10.8 was reached. The protein suspension was centrifuged for
15 min at 18000 g which gave four layers: a floating emulsion layer, a
clear supernatant, soft gel like sediment and harder bottom sediment.
The supernatant was separated from the emulsion layer by filtration
through a double layer cheese cloth. The solubilised proteins were
precipitated by adjusting the pH in the range of 4.8-7. The precipitated
proteins were centrifuged at 100000 g for 20 min. The results indicated
that the extract solubilised by acid had 92.1 ± 3.4% proteins compared
to 88.6 ± 3.8% solubilised by alkali.
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
Nurdiyana et al. [91] optimized the extraction process of proteins
from freeze dried fish waste using response surface methodology. The
fish waste obtained was minced in a blender and pre-treated with
petroleum ether to remove the fat. The defatted fish waste was freeze
dried for 24 h. The freeze dried fish waste was mixed with distilled water
in the ratio of 1:10 before adding NaOH. The results from the response
surface optimization methodology indicated that the optimum ratio of
NaOH:sample was 1.54:1, the optimum speed of rotation was 105 and
the optimum extraction time was 49 min at a pH of 10.5. The predicted
protein yield under these conditions was 85.02 mg/ml compared to an
experimental protein yield of 83.51 mg/ml.
Enzymatic extraction of fish protein
The enzymatic processing of various biopolymers in foodstuffs
(such as polysaccharides, proteins and pectins) is an important process
which is used to improve the physical, chemical and organoleptic
properties of the original food in relation to the nutritive value and
the intestinal absorption characteristics. The enzymatic extraction
of protein is carried out under controlled pH conditions without
degrading their nutritional qualities for the acceptance in the food
industry and broad spectrum of products can be produced for a wide
range of applications [92]. Many protein hydrolysates are subjected
to enzymatic processing to produce special diets for babies and sick
adults. This is possible only when the hydrolysates are low in bitterness,
osmotically balanced, hypoallergic and have good flavour. Most of
these diets are composed of peptides and are rich in amino acids [93].
Although enzymatic processing has been widely applied to various
livestock and poultry meat and milk, there are few studies on the
production of fish protein hydrolysate using enzymes. Fish processing
waste has also been underutilized for use as a feed or a fertilizer [46].
Most of the research studies conducted on the enzymatic processing of
fish protein seems to be laboratory or small scale oriented and have their
limitations when scaled up to industrial scale [94]. However, the large
scale production of fish protein hydrolysates are carried out in various
countries including France, Japan and other countries in Southeast
Asia. The process has several disadvantages including low yields, initial
high cost of enzymes, inactivation of enzymes after hydrolysis either by
heat or by pH and the inability to reuse enzymes [95].
The enzymatic processing of fish waste could be helpful in
producing a broad spectrum of food ingredients and industrial
products for a wide range of applications [96]. The enzymes used in the
food industry for the preparation of fish protein hydrolysate are mostly
carbohydrases, proteases and lipases.
Proteases are one of the most highly used enzymes in the food
industry [97]. Proteases are derived from animal, plant and microbial
Volume 5(4): 107-129 (2013) - 116
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
sources. Enzymes extracted from plant sources include papain,
bromelain and keratinases. Enzymes extracted from animal’s sources
include trypsin, chymotrypsin, pepsin and renin. Because of the inability
of plant and animal proteases to meet current demand in the market,
there is an increase in the demand for microbial proteases. Bacterial
proteases are often used in the production of protein hydrolysate
[46,98]. They are mainly neutral or alkaline and are produced by the
genus Bacillus. The neutral proteases are active in the pH range of 5-8
and have low temperature tolerance whereas the alkaline proteases are
active in the pH range of 7-10 and have broad specificity [98].
Alcalase is an alkaline enzyme produced from Bacillus licheniformis
which is developed by Novo Nordisk (Bagsvaerd, Denmark) for the
detergent industry. This enzyme has been proven to be one of the best
enzymes used to prepare fish protein hydrolysate [54,99]. Shahidi et al.
[92] stated that fish protein hydrolysate produced by alcalase had better
functional properties, a high protein content with an excellent nitrogen
yield, an amino acid composition comparable to that of muscle and a
higher nutritional value than those produced by other enzymes such
as Neutrase.
Liaset et al. [100] reported on the extraction of protein hydrolysate
from fish frames from Atlantic cod and Atlantic salmon using four
different enzymes neutrase, alcalase, pepsin and kojizyme. The study
revealed that after 120 min of hydrolysis, salmon treated with alcalase
and cod treated with pepsin yielded higher protein recoveries of 67.6%
and 64%, respectively.
Shahidi et al. [92] reported on the extraction of protein hydrolysate
from capelin (Mallotus villosus) using alcalase, neutrase and papain.
The samples were also subjected to autolytic hydrolysis. The results
revealed that protein recoveries with commercial enzymes reached
51.6-70% in comparison with the autolytic hydrolysis yield of 22.9%.
Alcalase hydrolysis had the highest protein recovery compared to those
other enzymes.
The effects of initial inactivation of endogenous enzymes, water
and different enzymes on the yield of proteins and oil from cod
(Gadusmorhua) were studied by Slizyte et al. [101]. The enzymes
used in the hydrolysis were alcalase and lecitase ultra. The results
revealed that initial heating of raw material changed its composition
and inactivated the endogenous enzymes. The yield of fish protein
hydrolysate had higher amount of lipids such as phospholipids and
other polar lipids. Alcalase with the addition of water produced good
quality fish protein and oil.
Guerard et al. [102] extracted protein from a yellowfin tuna
(Thunnus albacores) waste using alcalase. The freeze dried protein
hydrolysate was used as nitrogen substrate for microbial cultures such
as E. coli, L. casei, S. cerevisiae, S. odorus, P. roqueforti and A. niger.
Enzymatic hydrolysis was performed on the Atlantic spiny dogfish
(Squalus acanthias), which is of low commercial value but potential
source for high quality protein by Diniz and Martin [103]. The
optimized variables for protein extraction from dogfish waste were a
temperature of 35°C, a reaction time of 23.8 h, a rotation speed of 171
rpm and an enzyme: substrate ratio of 1.5. The protein yield was 80.75
g/L [91].
Beak and Cadwallader [104] reported on the extraction of
proteins from crayfish processing byproducts using alkaline protease
optimase. The optimal conditions for the enzymatic hydrolysis were
a temperature of 65°C, a pH of 8-9, a reaction time of 2.5 h and an
enzyme concentration of 0.3%. The maximum protein yield was 75%.
Utilization of Fish Protein
Fish protein contains many bioactive peptides that are easily
absorbed and can be used for various metabolic activities. They can
be used as a functional ingredient in many food items as they have
properties such as water holding capacity, oil absorption, gelling
activity, foaming capacity and emulsification property. Various
nutraceuticals are commercially produced and their applications are
shown in Table 15 [105,106]. Kristinsson and Rasco [46] reported
that fish protein hydrolysate can be used as a milk replacer with high
protein efficiency ratio (PER) value and in a cost effective manner
than dried skimmed milk. Due to its rich amino acid composition,
fish protein can be used as supplementing cereal proteins and can be
used in bakery products, soups and infant formulas. It is also reported
that fish proteins can serve as nitrogen source for the growth of micro
organisms for the production of extracellular lipases [106].
During the storage of fish under frozen conditions, impart
Product name
Preparation
Applications
Country
Seacure®
Prepared by hydrolyzing deep ocean white
fish proteins
Dietary supplement helps to support the cells in the gastrointestinal tract and regulate
bowel function.
US & Canada
Amizate®
Produced from Atlantic salmon fish proteins
by autolysis
Sports nutrition for muscle anabolism and metabolic recovery.
North America
Stabilium®200
Prepared from Molva dypterygia by autolysis Supports the body’s response to stress and provides nutritional support for memory and UK
cognitive function.
Protizen®
Produced by enzymatic hydrolysis of white
fish proteins
It is also called as “mood food” to fight against stress and symptoms such as weight
disorders, work pressure, sleep troubles and concentration difficulties
UK
Vasotensin®
Produced from Bonito (Sarda orientalis) by
thermolysin hydrolysis
It supports healthy vascular function for optimal blood flow and blood pressure vessel.
US and Japan
Peptace®
Produced from Bonito (Sarda orientalis) by
thermolysin hydrolysis
It lowers the blood pressure by inhibiting ACE enzyme.
US and Japan
Nutripeptin®
Manufactured by enzymatic hydrolysis of cod It helps in blood glucose stabilization and weight management.
fish fillet
Liquamen®
Prepared from Molva molva by autolysis
Dietary supplement that helps in reducing oxidative stress, lowering glycemic index and UK
anti stress
Molval®
Produced from North Atlantic fish Molva
molva by enzymatic hydrolysis
Dietary supplement recommended for cholesterol equilibrium, stress control and
promotes good cardiovascular health
UK
Seagest®
Prepared by hydrolysing deep ocean white
fish proteins
It supports the structure of the intestinal lining and health
US
UK and USA
Table 15: Commercial nutraceuticals from fish protein hydrolysates [106].
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
Volume 5(4): 107-129 (2013) - 117
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
changes on the texture and functional properties of fish proteins and
may eventually denature them. To prevent protein denaturation,
cryoprotectants are used to increase the surface tension and amount
of bound water to prevent ice formation and migration of water
molecules in the protein. Sugar-sorbitol in the ratio of 1:1 is commonly
used cryoprotectants. These fish products cannot be served for diabetic
patients due to the presence of carbohydrates in the cryoprotectants.
The protein hydrolysate from Pacific Hake, shrimp head can be used
as cryoprotectants without any loss in the cryoprotective property
when compared to other cryoprotectants [107-109]. The fish protein
hydrolysates also possess various bioactivities such as antioxidative,
antithrombic and antihypertensive properties.
Production and Utilization of Fish Amino Acids
Amino acids can be produced by hydrolyzing proteins. Chemical
(acid or alkali) and biological (enzymatic) methods are most
commonly used for the hydrolysis of proteins [46]. Microwave induced
hydrolysis of protein has also been reported. The aim of the hydrolysis
process is to liberate amino acids and recover them without degrading
their properties. The factors affecting the hydrolysis of proteins are
temperature, time, hydrolysis agent and additives. These factors affect
the quality and yield [110].
Acid hydrolysis of fish proteins
Acid hydrolysis is the most commonly used process for the
hydrolysis of proteins. The process itself is very harsh and hard to
control, but is still the preferred method for hydrolyzing proteins.
Acid hydrolysis is normally carried out using hydrochloric acid and
in some cases with sulfuric acid [111]. The agents used in the acidic
hydrolysis of proteins are shown in the Table 16. The conventional
acidic hydrolysis of fish proteins is carried out using 6 M HCl for 20-24
h at 110°C under vacuum [92]. Under these conditions of hydrolysis,
aspargine and glutamine are completely hydrolyzed to aspartic acid
and glutamic acid, respectively. Tryptophan is completely destroyed
and cysteine cannot be directly determined from the acid hydrolysed
samples. Tyrosine, serine and threonine are partially hydrolysed. There
is usually 5-10% loss in the recovery using acid hydrolysis [110].
The conventional method of acid hydrolysis was modified by
adding 50% acetic acid in order to reduce the hydrolysis time [112].
During conventional hydrolysis, the recoveries of amino acids are
very low and, therefore, in the presence of organic acid it is possible to
reach the hydrophobic regions of proteins. Tsugitaand Scheffler [113]
used formic acid, acetic acid, trifluoroacetic acid and propionic acid
to hydrolyze proteins. Trifluoroacetic acid was found to be a strong
acid with a pKa of 0.23, high vapour pressure and low boiling point
(72.5°C). The dipeptide consisting of valine and isoleucine (Val-Val,
Val-Ile, Ile-Val and Ile-Ile) in the proteins were hydrolyzed at 160°C for
25 min with various combinations of mixtures of hydrochloric acid and
organic acid. The results indicated that combination of trifluoroacetic
acid and HCl in the ratio of 1:1-1:2 showed a recovery of 100% when
compared to other organic acids as shown in Table 17.
Fountoulakis and Lahm [110] reported that the time and
temperature are always important variables to consider in conventional
acid hydrolysis processes. Hydrolysis of protein with 6 M HCl at 145°C
for 4 h gives comparable recoveries and quantification in comparison to
conventional hydrolysis with 6M HCl at 110°C for 24 h. The recoveries
of threonine and serine were reduced by 50% after 4 h, whereas the
valine and isoleucine recoveries increased by 100%. In comparison,
shortened hydrolysis at elevated temperatures gave similar or superior
results to those of conventional hydrolysis at 110°C for 24 h.
Csapo et al. [114] stated that in addition to the recovery yield, the
Hydrolysis agent
Hydrolysis Conditions
Additives
Specific
Determination
6 M HCl
110°C, 24 h
0.02% Phenol
All residues except of Cys, Trp
6 M HCl or 4 M MSA
110°C, 24 h
0.2% Sodium azide
Cys
6 M HCl
110°C, 18 h
5% Thioglycolic acid, 0.1% phenol, 3,3’-dithiodipropionic
acid
Cys
6 M HCl
3-Bromopropylamine
Cys
6 M HCl
145°C, 4 h
Samples previously oxidized with performic acid
Cys, Met, Lys
4 M MSA
3-(2-aminoethyl)indole
Trp, Methionine sulfoxide
4 M MSA
115°C, 22 h
Samples previously alkylated, tryptamine
All residues
4 M MSA
160°C, 45 min
All residues
4 M MSA or 5.7 M HCl
Oxidation with performic acid
150°C, 90 min
50°C, 10 min
All residues
3 M p-Toluenesulfonic acid
Methionine sulfoxide
12 M HCl–propionic acid (1:1)
150°C, 90 min
Resin-bound peptides
12 M HCl–propionic acid (1:1)
840 W, 1–7 min microwave
Resin-bound peptides
p-Toluenesulfonic acid
15 min, microwave
DCl
medium power, 30 min microwave Sensitive residues
2.5 M Mercaptoethanesulfonic acid
176°C, 12.5 min
S-Pyridylethylated samples
Cys, Trp
6 M HCl–TFA (6:3)
120°C, 16 h
Dithiodiglycolic acid, 1% phenol
Cys
HCl
Thioglycolic acid
Trp
HCl
110°C, 24 h
0.4% β- Mercaptoethanol
Trp
HCl
166°C, 25 min or 145°C, 4 h
3% Phenol
Trp
HCl
145°C, 4 h
Tryptamine
Trp
6 M HCl
145°C, 4 h gas phase
Tryptamine[3-(2-aminoethyl)]indole
Trp
TFA–HCl (1:2)
166°C, 25-50 min
5% Thioglycolic acid
Trp, Met
7 M HCl, 10% TFA
10% Thioglycolic acid, indole
Trp
Table 16: Hydrolysis agents of protein hydrolysis [110].
J Microb Biochem Technol
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Volume 5(4): 107-129 (2013) - 118
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
Acid Mixture
Formic acid: HCl
Acetic acid: HCl
Trifluoroacetic acid: HCl
Propionic acid: HCl
Composition (v/v)
Recovery (%)
1:1
85
1:2
95
1:1
97
1:2
100
2:1
85
1:1
100
1:2
100
1:1
90
1:2
97
Table 17: Recovery of amino acids from valyl- glutamic acid [113].
duration also affects the degree of racemization of the hydrolyzate.
When using conventional protein hydrolysis, racemization is 1.21.6 times higher compared to the hydrolysis carried out at elevated
temperatures of 160-180°C. At higher temperatures, proteins are
hydrolyzed rapidly into free amino acids and racemization of free amino
acids is always slower than that of amino acid bound to polypeptides.
During conventional acid hydrolysis the proteins are hydrolyzed at a
much slower rate, and the amino acids bound to polypeptide bonds
were exposed to heat for a longer time causing racemization. According
to Ozols [115], there are certain peptide bonds that are very tough to
cleave, including Ile-Val, Val-Val, Ile-Val, resulting in only 50-70%
yield at 110°C in 24 h and therefore hydrolysis must be carried out for
92-120 h in order to obtain higher yields.
Blackburn [116] used Methanesulfonic acid (MSA) in the presence
of 3-(2-aminoethyl) indole for the hydrolysis of proteins. The advantage
of using methane sulfonic acid in comparison to HCl is that tryptophan
is not destroyed and methionine is determined as methionine sulfoxide.
To reduce the losses incurred by the acid hydrolysis certain protective
agents such as phenol, thioglycolic acid, mercapto ethanol, indole or
tryptamine are added to the sample Adebiyi et al. [117] used a single
step protein hydrolysis process with 4 M methanesulfonic acid at 115°C
for 22 h in the presence of 0.02% tryptamine, in which even tryptophan
and cysteine were determined.
Chiou and Wang [118] hydrolyzed proteins using methane
sulfonic acid, in which 0.5 mg of protein samples were added with
0.5 ml 4 M methane sulfonic acid containing 0.2% 3-(2-aminoethyl)
indole. The hydrolysis tubes were flushed with nitrogen gas for 1 min,
closed tightly and heated at 160°C for 45 min. At the end of hydrolysis,
the samples were partially neutralized with 8 M sodium hydroxide to a
pH of 2. The samples were analyzed using amino acid analyzer and the
results were compared with conventional HCl hydrolysis. Hydrolyzing
the proteins at a higher temperature and a shorter time, accurate
results were produced for all amino acids including tryptophan and
half-cystine which are normally degraded in the conventional HCl
acid hydrolysis process. An additional advantage of this process is that
there is no degradation of serine, threonine and tyrosine as in the case
of prolonged hydrolysis. The amino acids recovery were between 97102% at a temperature of 160°C for 45 min. Methane sulfonic is also
non-volatile in nature and cannot be evaporated after hydrolysis and it
is, therefore, often neutralized to pH 2 before analysis.
Liu and Chang [119] hydrolyzed proteins using 3 N
p-toluenesulfonic acid containing 0.2% 3-(2-aminoethyl)indole at
110°C for 22, 48 and 72 h. At the end of hydrolysis, 2 ml of NaOH
were added and the mixture was transferred to 5 ml volumetric flask, in
which the total volume was made up to 5ml before analysis. The results
indicated that p-toluenesulfonic acid hydrolysis can be carried out to
J Microb Biochem Technol
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identify and quantify tryptophan but cannot be used for the analysis of
proteins which are contaminated with carbohydrates such as those in
animal feed.
Ault [120] stated that the extraction method is dependent on the
starting material which can include: hair, keratin, feather, blood meal
and soybeans. The standard procedure comprises of the hydrolysis with
aqueous acid, in which the amino acids are captured when passing the
hydrolysate over a strongly acidic ion exchange resin. The resin is later
washed with water and eluted with aqueous ammonia which frees
the amino acids. This is one of the most economical processes for the
production of tyrosine and cysteine. The main drawback of this process
is that the raw materials cannot keep up with the increasing demand for
amino acids [121].
Sano [122] reported on the extraction of amino acids from wheat
gluten which was identified as the major source of glutamate. The
process consists of three steps: extraction, isolation and purification.
In the extraction process, gluten was first separated from the wheat
flour by washing the starch from the dough. The crude gluten was
transferred to pottery vessels and mixed with hydrochloric acid and
heated for 20 h. The hydrolysates were then filtered to remove the black
residue resulting from the reaction of amino acids with carbohydrates.
The filtrate was transferred to the same vessel and concentrated for 24
h and then transferred to another vessel to crystallize for 1 month. The
crystals of L-glutamic acid hydrochloride were isolated from the liquid
through filtration and redissolved in water. The pH was adjusted to 3.2
and stored for 1 week for L-glutamic acid crystallization. The crystals
had two polymorphs granular α-form and stable, thin β-form. The
α-form contained only glutamate crystals with improved purity. The
separated L-glutamic acid α-form crystals were dissolved in water and
the pH was adjusted to 7 and filtered and decolorized using activated
charcoal. The filtered solution was concentrated by heating and cooled
to form monosodium L-glutamate.
Alkaline hydrolysis of fish protein
Hydrolysis of proteins can be carried out using sodium hydroxide,
potassium hydroxide or barium hydroxide. The alkaline treatment is
specifically used for the determination of tryptophan. It is also applied
to the samples which have a higher percentage of carbohydrates as in
the case of foods and formulation of pharmaceutical solutions which
have higher percentage of monosaccharides. The major disadvantage
of this method is that serine, threonine, arginine and cysteine are
destroyed and all other amino acids are racemized [123].
Linder et al. [124] stated that many deleterious reactions occur in
alkaline solutions during hydrolysis. These reactions are initiated by
hydrogen abstraction from the alpha carbon of an amino acid which
includes racemization of L- amino acids to produce D- amino acids.
Volume 5(4): 107-129 (2013) - 119
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
Lahl and Braun [125] reported that D- amino acids are not absorbed
by humans and that alkaline hydrolysis also splits disulfide bonds
with loss of cysteine, serine and threonine via β-elimination reactions
and formations of lysinoalanine, orinithinoalanine, lanthionine and
β-amino alanine. These eliminations can also lead to production of
toxic substances such as lysinoalanine and are undesirable in foods.
Bhumiratana et al. [131] studied the enzymatic solubilization of
fish protein in membrane reactors in batch, semi-batch and continuous
mode of operations using trypsin temperature of 50°C and pH of 9.0.
The results indicated that the fish proteins were completely solubilized
in the reactor and the functional properties of the product improved
better than the original substrate.
The chemicals that are produced during the alkaline hydrolysis
of protein are shown in the Figure 8 [46]. Protein phosphorylation
plays a major role in cell biology and biomedical sciences. Addition
of phosphate to the amino acid side chain by esterification causes
conformational changes to the protein in terms of activity and
stability. The typical acceptors of phosphate in their ring structure
are the hydrophobic amino acids such as tyrosine, serine, tryptophan,
histidine, aspartic acid, glutamic acid, lysine, arginine and cysteine.
For complete hydrolysis of the protein, a combination of different
proteases is necessary with a longer incubation time. Therefore, this
method is not applied for serial analysis [110]. Linder et al. [124]
stated that due to the presence of several peptide bonds and their
specific accessibility to enzymatic reaction, the enzymatic hydrolysis
is a complex process. The specificity of the enzymes is not the only
factor affecting the hydrolysis but some environmental factors such
as temperature and pH also play an important role in the hydrolysis
of the proteins. However, the widespread application of this type of
hydrolysis is hampered by the relative specificity of the proteases for
certain residues.
Yan et al. [126] reported the partial hydrolysis of amide bonds
using alkali can release phosphoamino acids. Fountoulakis and Lahm
[110] reported that potassium hydroxide hydrolysis was applied for
the quantification of phosphorylated and sulphated tyrosine and for
analysis of phosphohistidine. Proteolytic hydrolysis of protein samples,
followed by alkali hydrolysis results in superior yields of phosphoamino
acids [126].
Enzymatic hydrolysis of fish protein
Kim and Wijesekara [127] reported that enzymatic hydrolysis
of fish proteins with appropriate enzymes such as alcalase, pronase,
collagenase, pepsin, papain, protamex, bromelain, chymotrypsin
and trypsin allows for the preparation of bioactive peptides made up
of specific length of amino acids. The main advantage of enzymatic
hydrolysis of proteins is that it allows quantification of aspargine and
glutamine and other sensitive residues, which are normally destroyed
by acid and alkali hydrolysis, and does not cause any racemization
during digestion.
Wachirattanapongmetee et al. [128] hydrolysed fish proteins
obtained from hybrid Catfish frame. The proteins were mixed with
distilled water in the ratio of 1:1.5 (w/v) and the hydrolysis was started
by adding 0.5, 1.5 or 3% v/w protex enzyme. The fish proteins were
hydrolysed for 60, 120 and 180 min. The experiment was carried to
improve the functional properties of fish proteins such as protein
solubility, emulsification properties and fat absorption capacity rather
than completely breaking down the fish proteins into amino acids.
Cheftel et al. [129] hydrolysed fish protein using enzymes
(pronase, pepsin, bromein, ficin, rhozyme P11 and rhozyme 41) to
obtain complete solubilisation of proteins. The results indicated the
amount of protein hydrolyzed depended upon the type of enzyme
used and hydrolysis time. The rate constant of the reaction decreased
with increased with time and the hydrolysis did not follow first order
reaction. In addition, the formation of products within the system was
inhibitory to the rate of reaction. The results indicated that pronase
enzyme was able to hydrolyse 94% of the proteins in 23 h.
Yamashita et al. [130] hydrolysed fish protein concentrate (FPC)
in a two step hydrolysis process using two enzymes (papain and
pronase). The fish proteins were first hydrolysed using papain for 48
h at a temperature of 37°C and a pH of 1.5 with vigorous shaking.
The mixture was then hydrolysed using pronase at a temperature of
37°C for another 5 h at various pH conditions ranging from 6 to 9. The
protein was completely broken down and was comparable to that of
soy bean hydrolysates.
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To overcome the drawbacks of enzymatic hydrolysis of proteins,
enzyme membrane reactors (EMR) can be used to produce amino
acids. The set up for membrane reactors used for the production
of amino acids includes dead-end membrane; recycle membrane,
diffusion membrane and multiphase membrane reactors. In a deadend reactor, the solution containing enzymes is pushed towards the
membrane, the product is obtained on the other side and the enzymes
are retained (Figure 9a). In a recycle membrane reactor, the substrate
containing enzymes is continuously recycled from and to the reactor
through the filtration membrane (Figure 9b). If the soluble enzymes are
used, the reaction takes place in both vessel and membrane module but
occurs only in the module if immobilized enzyme are used [132,133].
The diffusion membrane reactor allows passive diffusion of substrate
molecules through the membrane to the adjacent compartment
containing enzymes (Figure 9c). These reactors are used only for low
molecular weight substrates (as the substrate is diffused back after
catalysis). The multi-phase reactors are capable of creating interfacial
contact between enzyme and substrate at the membrane matrix
through diffusion (Figure 9d). In this type of reactor, the membrane
acts as a support between two liquid phases and in some cases positive
pressure can be applied in order to prevent the phases from mixing
[133].
The main advantages of the enzyme membrane reactors (EMR)
include: (a) development of continuous processes for the production
of amino acids, (b) higher productivity, (c)controllable environment,
shift in the chemical equilibrium, (d) improved rate of reaction, (e)
concentration of process streams (f) possibility to conduct multiphase
process reactions, (g) no enzyme fixation costs, (h) interchange ability
of substrate/enzyme systems, (i) use of multienzyme system, (j)
Figure 8: Chemicals formed after alkaline hydrolysis [46].
Volume 5(4): 107-129 (2013) - 120
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
(PFA) pressure vessel which was free of the metal impurities and kept
in the microwave system at 125°C for 2-4 h. The results showed that
most of amino acids were completely hydrolyzed within 2 h except
valine, isoleucine and leucine which were resistant to hydrolysis.
Tryptophan was not stable and could not be measured. When the
time was increased to 4 h, threonine, serine and tyrosine were stable
compared to the conventional hydrolysis process.
Figure 9: Membrane Reactors [133].
sterilizability of the plant and (k) no diffusion limitation. The main
drawbacks of EMR are: (a) a specific substrate is used to produce
corresponding amino acids, (b) lacks operational stability, the enzymes
are resistant towards product inactivation, (c) high substrate and salt
concentrations, (d) it cannot withstand high temperature and organic
solvents, (e) it is not stable at low or high pH values, (f) poisoning of
enzyme, (g) deactivation of enzymes, (h) concentration polarization,
(i) fouling and (j) enzyme leakage [133,134].
Microwave induced hydrolysis of fish proteins
In the microwave induced hydrolysis of proteins, substrate can
be hydrolysed in either liquid or gas phase mode with HCl or other
reagents. In this method, the instant uptake of the radiation energy
results in the reduction of the overall hydrolysis time from many hours
to few minutes, 1-30 min for the liquid phase hydrolysis and 20-45
min for gas phase hydrolysis. There are no reports in the literature on
the use of this method with fish proteins. However, this method has
been used with bovine serum albumin, casein, gelatin and recombinant
human interferon α2 and has the potential for application in the fish
protein hydrolysis.
Englehart [135] reported that microwave hydrolysis of proteins
have been under investigation for determining the amount of amino
acids in connective tissues of meat products. The protein collagen in
the meat products contains a high concentration of amino acids which
can be determined by microwave hydrolysis.
Kaiser and Benner [136] studied microwave induced hydrolysis
of proteins (bovine serum albumin). Amino acids were hydrolysed
with a CEM Mars 5000 microwave equipped with a protein hydrolysis
accessory kit which included four Teflon vessels for the samples. The
major advantages of the process were: (a) it allows processing 40
samples in 3 h, (b) only 100 µl was needed for analysis, (c) the samples
contain only hydrochloric acid and moisture with less impurities and
(d) more feasible for smaller sized samples.
Margolis et al. [137] studied the hydrolysis of proteins (bovine
serum albumin) by microwave energy using bovine serum albumin
that was hydrolyzed with 10 mol/L HCl in a clean acid leached Teflon
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
Wu et al. [138] used microwave energy to study the cleavage of
synthetic peptide. In this study, synthetic peptides (1 mg/ml) were
dissolved in dilute hydrochloric acid (0.006, 0.015, 0.03, 0.06 M) and
water in 0.3 ml Teflon vials. The vials were flushed with nitrogen gas
for about min and placed in microwave oven for time intervals ranging
from 1 to 7 min. The results indicate that the peptides in the 0.06 M
HCl were completely hydrolyzed in 3 min and the peptides in the 0.03
M HCl were hydrolyzed in 4 min. The peptide content in the neutral
solution was less than 15% after 4 min. The microwave energy that is
emitted is absorbed into the liquid media by two mechanisms: ionic
conduction and dipole rotation. Therefore, the input power during
microwave hydrolysis plays an important role and 572-650 W power
is very suitable for microwave hydrolysis. This process was developed
to control the cleavage sites especially for the peptide bonds connected
to aspartic acid residues and it is very useful in obtaining defined acidcleaved peptide fragments.
Joergensen and Thestrup [139] performed microwave assisted
hydrolysis on protein samples (casein and gelatin). The results
suggested that a hydrolysis time of 10-30 min is enough to cleave all
the peptide linkages with no loss of serine or threonine. Methionine
was found to be stable with the addition of thioglycolic acid during the
hydrolysis process. As in the case of conventional hydrolysis process,
cysteine and tryptophan could not be quantified. To quantify cysteine,
the sample was subjected to pre-hydrolysis oxidation in which 20-50
mg of the protein were added to 5 ml ice cold performic acid (0.5 ml
30% H2O2 and 4.5 ml formic acid) and 250 µl of 200 m Mn or leucine
were also added along with 250 µl of 10% phenol at 0°C for 18 h. After
18 h, the reagents were dried under vacuum using a freeze dryer and
5 ml water and 10 ml 30% HCl were added to the dried samples to
perform microwave hydrolysis.
Weiss et al. [140] performed liquid phase and gas phase hydrolysis on
protein solutions (recombinant human interferon α2 from E. coli) using
a microwave technique. In liquid phase microwave hydrolysis, 500 µl
of protein solution were added to 6 M HCl containing 0.02% phenol or
to 4 M methane sulfonic acid containing 0.2% 3-(2-aminoethyl)indole.
The hydrolysis vial was placed in the microwave oven inside a beaker
containing 200 ml water for equal absorption of energy and hydrolysis
was carried out at 155°C (900 W) for 4 min. In the gas phase microwave
hydrolysis, 300 µl of protein solution were evaporated to dryness, 6 M
HCl were added and the hydrolysis was performed at 1000 W for 5
min and 500 W for 15 min after flushing the vials with argon gas. The
results indicated that using this microwave technique, the hydrolysis
was completed within 4 min and gave results comparable to that of
conventional hydrolysis with higher losses of serine and threonine and
a higher percentage of racemization of amino acids.
Utilization of amino acids
Amino acids are the building blocks of proteins. They have wide
nutritional value, taste, medicinal action and chemical properties. All
amino acids are sold in different quantities each year as shown in Table
18. They are used as food additives, in pharmaceutical applications,
feed and food supplements. The amino acids such as arginine, glycine,
Volume 5(4): 107-129 (2013) - 121
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
glutamate and histidine are used in protein pharmaceuticals as an
excipient for drug development is shown in Table 19. The largest
consumer of amino acids is the food flavoring industry which uses
monosodium glutamate, alanine, aspartate and arginine to improve
the flavour of food. The second largest consumer of amino acids is
the animal feed industry which uses lysine, methionine, threonine,
tryptophan and others to improve the nutritional quality of animal feed.
The amino acids can also used in various pharmaceutical applications
such as protein purification and formulations and production of
antibiotics such as jadomycin [141]. The total amino acid market in
1996 was estimated to be $4.5 billion. The market value of amino acids
has drastically increased since 1996 [121]. Fermentation products in
2004 were estimated to be $14.1 billion and $17.8 billion and amino
acids were the second most important category after antibiotics in 2009
[142].
Production and Utilization of Fish Oil
Chemical extraction of fish oil
Fish oil can be extracted from fish and fish-waste chemically using
Soxhlet or Goldfisch, Folch, Bligh and Dryer method and acid digestion
method with various solvents such as diethyl ether, petroleum ether,
chloroform/methanol and hexane [143].
Goldfisch method: In the Goldfisch method, 10 g of the predried
samples is placed in a ceramic extraction thimble. To the sample, 40 ml
of hexane or petroleum ether is added and kept for 4-7 h. The sample is
cooled and the solvent is evaporated at 95°C for 30 min. The sample is
cooled and the weight of the lipid is calculated as shown in Equations
1 and 2 [143-145].
Weight of lipid=(weight of container+extracted lipid)-(weight of
container) (1)
Lipid content (% ) =
Mass of lipid extracted (g)
× 100
weight of the original sample(g)
(2)
Hwang and Regenstein [146] reported the use of gold fisch
extraction procedure to determine the fat content of the frozen
menhaden fish at different temperatures and time intervals. Erickson
[147] carried out lipid extraction from catfish minced muscle using the
goldfisch method and compared it with other extraction methods of
Amino acid
Amount (ton/year)
Process
Uses
L- Glutamate
1000000
Fermentation
Flavour enhancer
D,L- Methionine
350000
Chemical
Food, Feed supplement and pharmaceutical
L- Lysine HCL
250000
Fermentation
Feed supplement
Glycine
22000
Chemical
Pharmaceutical, soy sauce
L- Phenylalanine
8000
Fermentation, synthesis
Aspartame
L- Aspartic acid
7000
Enzymatic
Aspartame, Pharmaceutical
L- Threonine
4000
Fermentation
Feed supplement
L- Cysteine
1500
Extraction, Enzymatic
Pharmaceutical
D, L- Alanine
1500
Chemical
Flavor, sweetener
L- Glutamine
1300
Fermentation
Pharmaceuticals
L- Arginine
1200
Fermentation
Flavor, Pharmaceuticals
L- Tryptophan
500
Fermentation, Enzymatic
Feed supplement, Pharmaceuticals
L- Valine
500
Fermentation
Pharmaceuticals
L- Leucine
500
Fermentation, extraction
Pharmaceuticals
L- Alanine
500
Enzymatic
Pharmaceuticals
L- Isoleucine
400
Fermentation
Pharmaceuticals
L- Histidine
400
Fermentation
Pharmaceuticals
L- Proline
350
Fermentation
Pharmaceuticals
L- Serine
200
Fermentation
Pharmaceuticals
L- Tyrosine
120
Extraction
Pharmaceuticals
Table 18: Global production of amino acids in 1996 [121].
Amino acids
Concentration
Product
Drug Substance
Company
L-Arginine
55 mg/mL
TNKase®
Human tissue plasminogen activator (tPA)
Genentech
L-Arginine
35 mg/mL
Activase®
Human tissue plasminogen activator (tPA)
Genentech
L-Glycine
21-25 mM
Kogenate®FS
Recombinant antihemophilic factor (rAHF)
Bayer
L-Glycine
3 mM
Synagis®
Human monoclonal antibody
MedImmune
L-Glycine
260 mM
BeneFIX®
Coagulation factor IX
Wyeth
L-Glycine
0.17-0.34 mg/mL
Nutropin®
Human growth hormone (hGH)
Genentech
Wyeth
L-Glycine
23 mg/mL
Neumega®
Interleukin-11
L- Glutamate
1 mg/mL
VariVax®
Varicella virus vaccine live
Merck
L-Glutamate
5 mg/mL
Streptase®
Streptokinase
Aventis
Baxter
L-Histidine
55 mM
Recombinate
Recombinate antihemophilic factor (rAHF)
L-Histidine
18-23 mM
Kogenate®FS
Recombinate antihemophilic factor (rAHF)
Bayer
L-Histidine
47 mM
Synagis®
Human monoclonal antibody
MedImmune
L-Histidine
5 mM
Herceptin®
Human monoclonal antibody
Genentech
L-Histidine
10 mM
BeneFIX®
Coagulation factor IX
Wyeth
Table 19: Approved protein pharmaceuticals containing amino acids as excipient [141].
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
Volume 5(4): 107-129 (2013) - 122
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
and found the chloroform/methanol method to be the better solvent
system for the lipid extraction from catfish.
Paredes and Baker [148] studied the physical, chemical and sensory
changes during thermal processing of canned catfish, ocean perch and
Atlantic Pollock. In this study, the fat from all the three species was
extracted using goldfisch extraction method.
Chloroform/Methanol method: Folch et al. [149] described a
chloroform/methanol lipid extraction procedure in which 1.5 g of fish
tissue was mixed with 30 mL of 2:1 chloroform/methanol. The mixture
was then filtered and washed several times using 2:1 chloroform/
methanol and a weak salt solution of 0.88% NaCl was then added
to get a final ratio of 8:4:3 chloroform/methanol/water. The final
biphasic layer was centrifuged ant the lower phase was collected in a
pre-weighed glass tube and evaporated to dryness under nitrogen in
a thermostatically controlled water bath maintained at 25–30°C. To
completely remove all final traces of solvent and water, the sample
tube was then flushed with nitrogen and vacuum suction was applied
for 5 min. The lipid content was then determined gravimetrically by
weighing the remaining lipids in the tube.
Iverson et al. [150] extracted lipids from fish samples using the
original procedure described by Folch et al. [149] and the results were
compared to Bligh and Dyer method of lipid extraction. The results
suggested that both methods of extractions were highly correlated
and the only advantage of using Bligh and Dyer is the reduction in
the solvent/sample ratio; 1 part of tissue to 3 parts in the chloroform/
methanol method instead of 1 part of tissue to 20 parts of chloroform/
methanol in the Folch method.
Bell et al. [151] extracted lipids from the Atlantic salmon
(Salmosalar) using the procedure described by Folch et al. [149].
Gigliotti et al. [152] reported on the extraction of lipids from Atlantic
krill (Euphausiasuperba) using Folch method. Ramanathan and Das
[153] extracted lipids from Scomberomorus commersoni using Folch
method to study the lipid oxidation of the ground fish in the presence
of antioxidants. Bernardez et al. [154] reported on the extraction of
lipids from Atlantic mackerel (Scomberscombrus) using Folch method
and analyzed the free fatty acid in the fish. Saify et al. [155] extracted
lipids from two species of shark using chloroform/methanol (2:1)
extraction method (Folch method) and evaluate lipid composition of
both the species.
Bligh and dyer: In the Bligh and Dyer [156] method, 100 g of sample
is homogenized with 100 ml of chloroform and 200 ml methanol. To
this mixture, 100 ml of chloroform is added and homogenised for 30
seconds. Then 100 ml of distilled water or weak salt solution (0.88%
NaCl) is added to the mixture. The homogenate is filtered using
Whattman No. 1 filter paper. The filtrate is then transferred to 500 ml
measuring cylinder and allowed for separation and clarification. The
chloroform later contains the lipid which is then separated through
distillation. Lipid content was then determined gravimetrically by
weighing the remaining lipids in the tube.
Iverson et al. [150] extracted lipids from fish tissues using the Bligh
and Dyer extraction method. Smedes and Askland [157] extracted
lipids from cod fillet using Bligh and Dryer method and determined the
total lipid content in the fish. Norziah et al. [158] extracted fish oil from
wastes of seafood processing industry using Bligh and Dyer method
and determined the peroxide and anisidine values.
Ozogul et al. [159] compared the petroleum ether solvent extraction
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and Bligh and Dyer extraction for changes in the EPA and DHA. The
results indicated that that Bligh and Dyer method was more efficient in
extracting polar and non-polar lipids than the petroleum ether method
and also prevented losses of polyunsaturated fatty acids by reduction
in oxidation.
Aryee and Simpson [160] extracted fish oil from salmon skin using
Bligh and Dyer method and determined the total yield. Gruger et al.
[161] extracted lipids from 21 species of marine fish, freshwater fish
and shellfish using Bligh and Dyer method and determined the fatty
acid composition of fish species. Indrati et al. [162] extracted fatty
acids from fish oil and cod liver oil using Bligh and Dyer method and
determined the fatty acid methyl esters (FAME) present in the oil.
Zhong et al. [163] extracted lipids from the muscles and viscera
of steelhead trout using Bligh and Dyer method with a chloroform/
methanol ratio of 2:1 to characterize the fatty acid composition of
muscles and viscera. The results indicated that muscles had more
polyunsaturated fatty acids than viscera.
Acid digestion method: In the acid digestion method, 5 g of
ground sample is hydrolysed using 6 N HCl at 80°C for 1 h or at 110°C
for 4-24 min until complete dissolution. The lipids are extracted using
1:1 (v/v) chloroform/methanol, retaining the organic layers each time.
The organic solvent is removed at 40°C under reduced pressure using
arotary evaporator. Then, the weight and content of lipid are calculated
[143].
Xiao [144] extracted lipids using acid digestion method, in which
4 g of fish sample was weighed in a flask and dried at 103°C in an oven
for one hour. After drying, 100 ml of petroleum ether were to the added
to the flask and extraction was carried out for 2 h. The petroleum ether
was then distilled and the sample was again heated for 1 h at 103°C.
Then, 3 ml of hydrochloric acid were added and the sample was boiled
for 1 h in a water bath. The sample was cooled and filtered. The filtrate
was washed with distilled until the filtrate is neutral. The sample was
again dried for 1 h at 103°C. 100 ml of petroleum ether were again
added to the sample and extracted overnight with a condensation rate
of approximately 2-3 drops/second. The petroleum ether was distilled
and the sample was dried in oven for 2 h at 103°C. The fat content was
calculated as follows:
=
Fat
a−b
× 100 w
(3)
Where,
a=Weight of the tube with fat extract (g)
b=Weight of the tube (g)
w=Initial weight of the sample (g)
Radar et al. [164] determined the total fat and saturated fat in
various food stuffs including fish using gas chromatography after acid
hydrolysis using 6N hydrochloric acid and petroleum ether.
Christie [165] stated that during extraction of lipids, problems may
occur due to the presence of highly hydrophilic functional groups such as
acyl-coenzyme A esters, lysophospholipids and polyphosphoinositides
and gangliosides. Especially with polyphosphoinositides, it always
necessary to store the fish tissues in a proper condition, so that enzymatic
degradation is minimized. In such cases, the tissues are extracted with
chloroform/methanol in the presence of calcium chloride initially and
with 1 M hydrochloric acid for better recovery of lipids.
Volume 5(4): 107-129 (2013) - 123
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
Enzymatic extraction of fish oil
The enzymatic hydrolysis of fish is carried out using various
commercially available enzymes such as alcalase, neutrase, lecitase
ultra, protex and protamex. In the enzymatic extraction method, the
fish samples are minced and 50 g of fish mince is initially heated at 90°C
for 5 min to deactivate endogenous enzymes and to the heated samples
50 ml of water or buffer is added. The temperature and pH of the fish
mince is adjusted to 55°C and 8.0 for alcalase, 45°C and 6.5 for neutrase,
40°C and 6.8 for lecitase ultra, 60°C and 9.0 for protex and 50°C and 7.5
for protamex. The hydrolysis is started by adding 0.5% (w/w) enzyme
to the mixture and proceeded for 1, 2, 3 or 4 h. After each hydrolysis
time intervals the samples are heated at 90°C for 5 min to deactivate the
added enzymes. The mixture is then centrifuged at 10000 rpm for 20
min to separate oil fraction from the mixture [166-169].
Mbatia et al. [170] used 0.5% bromelain and 0.5% protex to extract
oil from nile perch and salmon heads at 55°C. The results indicated that
a maximum oil yield of 11.6 g/100 g and 15.7 g/100 g was achieved for
bromelain and protex, respectively. The oil recovered from the available
total lipids in salmon heads and nile perch using bromelain and protex
were 65% and 88% and 81% and 81%, respectively. The study also
suggested that increasing the enzyme concentration increased the
hydrolysis rate but did not increase the oil yield from fish.
Linder et al. [171] used three different enzymes Neutrase, Alcalase
and Flavourzyme at a concentration of 0.05% and three temperatures
(45, 55 and 50°C) for 2 h to extract oil from salmon heads. The oil yield
using Neutrase, Flavourzyme and Alcalase were 17.2, 17.0 and 17.4%,
respectively.
Dauskas et al. [172] extracted oil from cod (Gadusmorhua) by
products using alcalase enzyme at 50°C and pH of 6.5 from various
parts of cod fish. The study reported maximum oil recovery of 82.8%
from cod viscera without digestive tract using flavourzyme enzyme.
The lowest oil recovery of 36.4% was achieved by using Neutrase
enzyme on viscera with backbone. The authors suggested that at the
end of hydrolysis lipids were formed in three forms: free oil, emulsion
and sludge. The formation of emulsion is not desirable and increase
in the amount of emulsion decreases the amount of free oil produced.
The study suggested that addition of water during hydrolysis increased
the formation of emulsion and decreased the production of free oil. In
this study the highest oil yield (76.26%) was achieved from head and it
was less than the oil yield reported by the author which is due to the
addition of buffer during the hydrolysis process.
Slizyte et al. [167] extracted oil from cod (Gadusmorhua) by
products using alcalase enzyme at 55°C and pH of 7.5 and lecitase
ultra at 70°C and pH of 8.5 for 60 min. In this study, the extraction
was carried out by varying parameters such as heat inactivation and
addition of water both individually and in combination of enzymes.
The oil is obtained by centrifugation at 10000 g for 10 min. The
results indicated that the alcalase treated samples without addition of
water gave best results followed by the combination of alcalase and
lecitase without addition of water. The addition of water increased the
emulsion formation and increased the protein yield and decreased the
oil yield. The heat inactivation prior to start of the hydrolysis increased
the amount of oil released into the system.
Slizyte et al. [101] used flavourzyme and Neutrase to extract oil
from cod and reported that the decrease in the amount of free oil
fraction can be attributed to the presence of large amounts of proteins
J Microb Biochem Technol
ISSN: 1948-5948 JMBT, an open access journal
in the raw material (digestive tracts, flesh and backbones) which
together with the oil present in the liver forms various complexes when
heated during thermal deactivation of endogenous enzymes. During
heat inactivation, the proteins in the raw material were denatured
and precipitated. Only a small portion of denatured proteins can be
solubilised and the remaining forms a lipid-protein complex which
eventually reduces the release of lipids into the oil fraction. The report
also suggested that the minimum amount of lipids in the raw material
should be more than 8.5 g/100 g to form an emulsion and to decrease
the formation of emulsion the amount of protein must be higher than
16.5 g/100 g.
Utilization of Fish Oil
For human consumption
Fish oils are readily available sources for long chain polyunsaturated
fatty acids whichconsist of omega-3 fatty acids mainly composed of
cis-5,8,11,14,17- eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19docosahexaenoic acid (DHA) [173]. The omega-3 fatty acids have
many beneficial bioactivities including prevention of atherosclerosis,
protection against arrhythmias, reduced blood pressure, benefit to
diabetic patients, protection against manic-depressive illness, reduced
symptoms in asthma patients, protection against chronic obstructive
pulmonary diseases, alleviating the symptoms of cystic fibrosis,
improving survival of cancer patients, reduction in cardiovascular
disease and improved learning ability [96,156,174]. The American
Heart Association has recommended at least two servings of fish every
weekto reduce the effect of cardiovascular diseases [175].
For biodiesel production
Biodiesel is comprised of monoalkyl esters of vegetable oils, animal
fats or fish oils which can be synthesized from edible, non-edible and
waste oils. It is a non-toxic, biodegradable and renewable energy source.
Biodiesel can be produced chemically or enzymatically. Currently,
the biodiesel production on an industrial scale is being carried out
chemically, using alkali (NaOH) as catalyst due to high conversion
ratio of triglycerols (TAG) to methyl esters (biodiesel) and low reaction
times (4-10 h). [176]. There are several disadvantages using chemical
catalysts including high reaction temperature, soap formation, waste
generation and contamination of glycerol with alkali catalysts.
The biodiesel production using enzymatic transesterification
is mainly carried out using lipases such as Canadida Antarctica,
Carica papaya, Rhizopusoryzae, Pseudomonas cepacia, Pseusdomonas
fluoresces, Rhizomucormiehei and Mucormiehei. The advantages
of using enzymatic method are: there is no soap formation, low
temperature requirement, no waste generation and high quality of
glycerol. The disadvantages of using enzymatic transesterification are:
high reaction times (12-24 h) and high cost of enzymes [176-178].
Conclusion
Fish processing is one of the major industries in Canada. Processing
of fish involves stunning, grading, slime removal, deheading, washing,
scaling, gutting, cutting of fins, meat bone separation and steaks and
fillets. During these steps significant amount of waste is generated
which can be utilized as fish silage, fishmeal and fish sauce. Fish waste
can also be used for production of various value added products such
as proteins, oil, amino acids, minerals, enzymes, bioactive peptides,
collagen and gelatin.
Volume 5(4): 107-129 (2013) - 124
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
The fish proteins are found in all parts of the fish. There are three
types of proteins in fish: structural proteins, sacroplasmic proteins
and connective tissue proteins. The fish proteins can be extracted by
chemical and enzymatic process. In the chemical method, salts (NaCl
and LiCl) and solvents (isopropanol and aezotropic isopropanol) are
used, whereas during the enzymatic extraction, enzymes (alcalase,
neutrase, protex, protemax and flavorzyme) are used to extract proteins
from fish. These fish proteins can be used as a functional ingredient in
many food items because of their properties (water holding capacity,
oil absorption, gelling activity, foaming capacity and emulsifying
properties). They can also be used as milk replacers, bakery substitutes,
soups and infant formulas.
The amino acids are the building blocks of protein. There are 16-18
amino acids present in fish proteins. The amino acids can be produced
from fish protein by enzymatic or chemical processes. The enzymatic
hydrolysis involves the use of direct protein substrates and enzymes
such as alcalase, neutrase, carboxypeptidase, chymotrypsin, pepsin and
trypsin. In the chemical hydrolysis process, acid or alkali is used for the
breakdown of protein to extract amino acids. The main disadvantage of
this method is the complete destruction of tryptophan and cysteine and
partial destruction of tyrosine, serine and threonine. The amino acids
present in the fish can be utilized in animal feed in the form of fishmeal
and sauce or can be used in the production of various pharmaceuticals.
The fish oil contains two important polyunsaturated fatty acids
called EPA and DHA or otherwise called as omega-3 fatty acids. These
omega-3 fatty acids have beneficial bioactivities including prevention
of atherosclerosis, protection against maniac–depressive illness and
various other medicinal properties. Fish oil can also be converted
to non-toxic, biodegradable, environment friendly biodiesel using
chemical or enzymatic transesterification.
Acknowledgement
The research was supported by the Natural Sciences and Engineering
Research Council (NSERC) of Canada.
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Volume 5(4): 107-129 (2013) - 128
Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D (2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129. doi:10.4172/1948-5948.1000110
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Citation: Ghaly AE, Ramakrishnan VV, Brooks MS, Budge SM, Dave D
(2013) Fish Processing Wastes as a Potential Source of Proteins, Amino
Acids and Oils: A Critical Review. J Microb Biochem Technol 5: 107-129.
doi:10.4172/1948-5948.1000110
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